The present invention relates to novel DNAs which are useful as DNA primers for a polymerase chain reaction (PCR); methods for amplifying DNAs each coding for a G protein coupled receptor protein via PCR techniques using said DNA; screening methods for DNAs each encoding a G protein coupled receptor protein via PCR techniques using said DNA; G protein coupled receptor protein-encoding DNAs obtained by said screening method; G protein coupled receptor proteins which are encoded by the DNA obtained via said screening method, peptide fragments or segments thereof, and modified peptide derivatives thereof; etc.
The present invention also relates to novel G protein coupled receptor proteins; novel G protein coupled receptor protein-encoding DNAs; processes for producing said G protein coupled receptor protein; use of said receptor protein and said protein-encoding DNA; etc.
The present invention also relates to novel human amygdaloid nucleus-derived G protein coupled receptor proteins;
novel DNAs each coding for said G protein coupled receptor protein; processes for producing said G protein coupled receptor protein; use of said receptor protein and said protein-encoding DNA; etc.
The present invention also relates to novel mouse pancreatic xcex2 cell line MIN6-derived G protein coupled receptor proteins; novel DNAs each coding for said G protein coupled receptor protein; processes for producing said G protein coupled receptor protein; use of said receptor protein and aid protein-encoding DNA; etc. Further, the present invention relates to novel human-derived G protein coupled receptor proteins (human prinoceptors); novel DNAs each coding for said G protein coupled receptor protein; processes for producing said G protein coupled receptor protein; use of said receptor protein and said protein-encoding DNA; etc.
A variety of hormones, neurotransmitters and the like control, regulate or adjust the functions of living bodies via specific receptors located in cell membranes. Many of these receptors mediate the transmission of intracellular signals via activation of guanine nucleotide-binding proteins (hereinafter, sometimes referred to as G proteins) with which the receptor is coupled and possess the common (homologous) structure, i.e. seven transmembranes (membrane-spanning regions (domains)). Therefore, such receptors are generically referred to as G protein coupled receptors or seven transmembrane (membrane-spanning) receptors.
G protein coupled receptor proteins have a very important role as targets for molecules such as hormones, neurotransmitters and physiologically active substances, which molecules control, regulate or adjust the functions of living bodies. Each molecule has its own receptor protein which is specific thereto, whereby the specificities of individual physiologically active substances, including specific target cells and organs, specific pharmacological actions, specific action strength, action time, etc., are decided. Accordingly, it has been believed that, if G protein coupled receptor genes or cDNA can be cloned, those will be helpful not only for the clarification of structure, function, physiological action, etc. of the G protein coupled receptor but also for the development of pharmaceuticals by investigating the substances which act on the receptor. Until now, only several G protein coupled receptor genes or cDNAs have been cloned but it is believed that there are many unknown G protein coupled receptor genes which have not been recognized yet.
The characteristic feature of the G protein coupled receptor proteins which have been known up to now is that seven clusters of hydrophobic amino acid residues are located in the primary structure and pass through (span) the cell membrane at each region thereof. It has been known that such a structure is common among all of the known G protein coupled receptor proteins and further that the amino acid sequences corresponding to the area where the protein passes through the membrane (membrane-spanning region or transmembrane region) and the amino acid sequences near the membrane-spanning region are often highly conserved among the receptors. When an unknown protein has such a structure, it is strongly suggested that said protein is within a category of the G protein coupled receptor proteins. In addition, some amino acid residue alinements are common (homologous) and, by taking it as a characteristic feature, it is further strongly suggested that said protein is a G protein coupled receptor protein.
Libert, F, et al. (Science, 244:569-571; 1989) reported a method for cloning novel receptor genes by means of a polymerase chain reaction (hereinafter, sometimes referred to as PCR or a PCR technique) for a synthetic DNA primer which was synthesized based upon the information of common amino acid sequences obtained from a comparison among known G protein coupled receptor proteins. Libert, F. et al. used a pair of synthetic DNA primers corresponding to the portions of the third and the sixth membrane-spanning regions. However, in general, the design of primers used for the PCR regulates the molecular species of DNAs which are to be amplified. In addition, when a similarity (homology) in the amino acid sequence level is used as a basis, the use of different codons affects on the binding (hybridization) of the primer thereby resulting in a decrease in the amplifying efficiency. Accordingly, although various novel receptor protein DNAs have been obtained using said DNA primers, it is not possible to succeed in amplifying DNAs for all receptor proteins in the prior art.
Further, the amino acid sequence which is common to from the first to the seventh membrane-spanning regions among 74 G protein coupled receptor proteins was reported by William C. Probst, et al. (DNA and Cell Biology, Vol. 11, No. 1, 1992, pp. 1-20). In this report, however, there is no suggestion for a method in which DNA coding for a novel G protein coupled receptor protein is screened by means of PCR using DNA primers which are complementary to the DNA coding for those amino acid sequences.
It would be desirable to develop DNA primers for PCR techniques which allow selective and efficient screenings of DNAs coding for the areas (regions) more nearer the full length of novel G protein coupled receptor proteins by utilizing the common (homologous) sequence(s) of the G protein coupled receptor protein or the DNA coding therefor.
It would also be desirable to develop synthetic DNA primers corresponding to the portions of the third and the sixth membrane-spanning regions, said primer being useful in screening for DNA coding for G protein coupled receptor proteins in more selective and efficient manner as compared with a series of the synthetic DNA primers corresponding to the sequences of the third to the sixth membrane-spanning regions as reported by Libert, F. et al.
G protein coupled receptor proteins are important for investigating substances which control the function of living organisms and proceeding developments thereof as pharmaceuticals. Finding and development of candidate compounds for new pharmaceuticals can be efficiently proceeded by using G protein coupled receptor proteins and by conducting receptor binding experiments and evaluating experiments on agonists/antagonists using intracellular information transmittance systems as indexes. Especially when the presence of a novel G protein coupled receptor protein can be clarified, the presence of a substance having a specific action thereon can be suggested.
If a novel DNA which codes for a novel G protein coupled receptor protein can be efficiently screened and isolated, it will now be possible to proceed with the isolation of DNA having an entire coding region, the construction of an expression system therefor and the screening of an acting ligand.
A hypothalamo-hypophysial system is one of the passages for controlling, regulating or adjusting the functions of organisms relying upon interactions of hormones and neurotransmitters with G protein coupled receptors. In the hypothalamo-hypophysial system, the secretion of pituitary hormones from the pituitary body (hypophysis) is regulated by hypothalamic hormones (hypophysiotropic releasing factors), and the functions of target cells and organs are controlled by pituitary hormones released into the blood. Functions which are important for the living body are regulated through this system, such as maintenance of homeostasis and control of development and growth of a genital system and an individual organism. Representative examples of the hypothalamic hormones include TRH, LH-RH, CRF, GRF, somatostatin, galanin, etc. Representative examples of the pituitary hormones include TSH, ACTH, FSH, LH, prolactin, growth hormone, oxytocin, vasopressin, etc. In particular, the secretion of pituitary hormones is regulated according to a positive feedback mechanism or a negative feedback mechanism relied on the hypothalamic hormones and peripheral hormones secreted from the target endocrine glands. A variety of receptor proteins present in the pituitary gland play a major role for regulating the hypothalamo-hypophysial system.
It has been widely known that these hormones, factors and receptors are widely distributed in the brain instead of existing only locally in the hypothalamo-hypophysial system. This fact suggests that the substances which are called xe2x80x9chypothalamic hormonesxe2x80x9d are working as neurotransmitters or neuroregulators in the central nervous system. It is further considered that these substances are similarly distributed even in the peripheral tissues to play the role of important functions. The pancreas plays an important role of carrying out the carbohydrate metabolism by secreting not only a digestive fluid but also glucagon and insulin. Insulin is secreted from the xcex2 cells and its secretion is promoted chiefly by glucose. It has, however, been known that a variety of receptors exist in the xcex2 cells, and the secretion of insulin is controlled by various factors such as peptide hormones (galanin, somatostatin, gastric inhibitory polypeptide, glucagon, amylin, etc.), sugars (mannose, etc.), amino acids, and neurotransmitters in addition to glucose.
It has thus been known that in the pituitary gland and in the pancreas are present receptor proteins for many hormones and neurotransmitters, said receptor proteins playing important roles for regulating the functions. As for the galanin and amylin, however, there has not yet been reported any discovery concerning the structure of their receptor protein cDNAs. It is not known whether there exist any unknown receptor proteins or receptor protein subtypes.
For substances regulating the functions of the pituitary gland and pancreas, there exist receptor proteins specific to said substance on the surfaces of various functional cells of the pituitary gland and pancreas. The pituitary gland and the pancreas are associations of a plurality of functional cells, and the actions of the individual substances are defined by the distributions of their target receptor proteins among the functional cells. Accordingly, a substance, in many cases, exhibits an extensive variety of actions. To comprehend such complex systems, it is necessary to clarify the relations between the acting substances and the specific receptor proteins. It is further necessary to efficiently screen for receptor protein agonists and antagonists capable of regulating the pituitary gland and pancreas, to clarify the structures of genes of receptor proteins from the standpoint of investigating and developing pharmaceuticals, and further to express them in a suitable expression system.
By utilizing the fact that a G protein coupled receptor protein exhibits homology in part of the structure thereof at the amino acid sequence level, an experiment of looking at DNAs coding for novel receptor proteins relying upon a polymerase chain reaction (hereinafter simply referred to as xe2x80x9cPCRxe2x80x9d) has recently been made.
In the central nervous system, many receptor proteins such as dopamine receptor protein, LH-RH receptor protein, neurotensin receptor protein, opioid receptor protein, CRF receptor protein, CRF receptor protein, somatostatin receptor protein, galanin receptor protein, TRH receptor protein, etc. are G protein coupled receptor proteins, and it has been clarified that ligands to these receptors exert a variety of effects in the central nervous system.
In the immune system, an xcex1- or a xcex2-chemokine receptor protein, an MIPIxcex1 receptor protein, an IL-8 receptor protein, a C5a receptor protein, etc. have been known as such G protein coupled receptor proteins, and are working as receptor proteins responsive to immunoregulating substances to play important roles for regulating the functions of the living body. There is, for example, an IL-6 receptor protein that acts both in the above-mentioned central nervous system and in the immune system. IL-6 is both a xcex2-cell differentiating factor and a biologically active factor related to the proliferation and differentiation of nerve cells.
It has been widely known that these hormones, factors and receptor proteins are usually widely distributed up to the peripheral tissues instead of existing only locally in the central nervous system and in the immune system and are producing important functions, respectively. Agonists and antagonists for these receptor proteins are now being developed as various useful pharmaceuticals.
For substances regulating the functions of the central nervous system and the immune system, there exist receptor proteins specific to said substance on the surfaces of various functional cells of the central nervous system and the immune system. The central nervous system and the immune system are associations of a plurality of functional cells, and the actions of the individual substances are defined by the distributions of their target receptor proteins among the functional cells. Accordingly, a substance, in many cases, exhibits an extensive variety of actions. Moreover, there is an example wherein many factors play a part in a physiological phenomenon. To comprehend such complex systems, it is necessary to clarify relations between the acting substances and the specific receptor proteins.
As discussed herein above, the G protein coupled receptor protein is present on the cell surface of living body cells and organs and has a very important role as a target for molecules such as hormones, neurotransmitters and physiologically active substances, which molecules control, regulate or adjust the functions of living body cells and organs.
One object of the present invention is to provide novel DNAs which are useful as DNA primers for a polymerase chain reaction; methods for amplifying a DNA coding for a G protein coupled receptor protein using said DNA; screening methods for the DNA coding for a G protein coupled receptor protein using said DNA; DNAs obtained by said screening method; and G protein coupled receptor proteins encoded by the DNA obtained by said screening method, peptide fragments or segments thereof, modified peptide derivatives thereof or salts thereof.
Another object of the present invention is to provide processes for producing said receptor protein; transformants capable of expressing said receptor protein; cell membrane fractions obtained from said transformant; methods for determining a ligand to the receptor protein; screening methods for a compound or a salt thereof capable of inhibiting the binding of the ligand with the receptor protein; kits for said screening method, pharmaceutical compositions comprising an effective amount of the inhibitory compound; antibodies against said receptor protein; immunoassays using said receptor protein or said antibody and use of said receptor protein and encoding DNA.
Yet another object of the present invention is to provide novel G protein coupled receptor proteins which are expressed in pituitary glands or pancreatic xcex2 cells; DNAs comprising a DNA coding for said G protein coupled receptor protein; processes for producing said receptor protein; transformants capable of expressing said receptor protein; cell membrane fractions obtained from said transformant; methods for determining a ligand to the receptor protein; screening methods for a compound or a salt thereof capable of inhibiting the binding of the ligand with the receptor protein; kits for said screening method, pharmaceutical compositions comprising the inhibitory compound; antibodies against said receptor protein; immunoassays using said receptor protein or said antibody and use of said receptor protein and encoding DNA.
Still another object of the present invention is to provide novel human amygdaloid nucleus-derived G protein coupled receptor proteins; DNAs comprising a DNA coding for said G protein coupled receptor protein; processes for producing said receptor protein; transformants capable of expressing said receptor protein; cell membrane fractions obtained from said transformant; methods for determining a ligand to the receptor protein; screening methods for a compound or a salt thereof capable of inhibiting the binding of the ligand with the receptor protein; kits for said screening method, pharmaceutical compositions comprising the inhibitory compound; antibodies against said receptor protein; immunoassays using said receptor protein or said antibody and use of said receptor protein and encoding DNA.
Yet another object of the present invention is to provide novel mouse pancreatic xcex2 cell line MIN6-derived G protein coupled receptor proteins; DNAs comprising a DNA coding for said G protein coupled receptor protein; processes for producing said receptor protein; transformants capable of expressing said receptor protein; cell membrane fractions obtained from said transformant; methods for determining a ligand to the receptor protein; screening methods for a compound or a salt thereof capable of inhibiting the binding of the ligand with the receptor protein; kits for said screening method, pharmaceutical compositions comprising the inhibitory compound; antibodies against said receptor protein; immunoassays using said receptor protein or said antibody and use of said receptor protein and encoding DNA.
The present inventors have succeeded in synthesizing novel DNA primers based upon the similarity (homology) with the base sequences coding for the first membrane-spanning region or the sixth membrane-spanning region each of known G protein coupled receptor proteins. It is to be particularly noted that there has been no report of a DNA primer pair which has been synthesized paying attention to the similarity with the base sequence coding for the first and the sixth membrane-spanning region of the known G protein coupled receptor protein.
Next the present inventors have succeeded in synthesizing other novel DNA primers based upon the similarity (homology) with the base sequences coding for the third or the sixth membrane-spanning region each of known G protein coupled receptor proteins. They have also unexpectedly succeeded in efficiently amplifying DNAs (DNA fragments) coding for G protein coupled receptor proteins by means of PCR using those DNA primers.
They have further succeeded in synthesizing novel DNA primers based upon the similarity (homology) with the base sequences coding for the second or the seventh membrane-spanning region each of known G protein coupled receptor proteins; upon the similarity (homology) with the base sequences coding for first or the third membrane-spanning region each of known G protein coupled receptor proteins; and upon the similarity (homology) with the base sequences coding for the second or the sixth membrane-spanning region each of known G protein coupled receptor proteins. They have furthermore and unexpectedly succeeded in efficiently amplifying DNAs (DNA fragments) coding for G protein coupled receptor proteins by conducting PCR using those DNA primers.
Moreover, the present inventors have succeeded in efficiently cloning full-length DNA coding for said G protein coupled receptor protein via using amplified DNAs (DNA fragments) coding for said G protein coupled receptor protein. Thus, they have found that novel DNA coding for novel G protein coupled receptor proteins can be isolated, characterized or prepared via conducting amplifications and analyses of various DNA using said DNA primers.
To be more specific, the present inventors have selected amino acid sequences which are each common to the portion corresponding to or near the first and the sixth membrane-spanning region of the known individual G protein coupled receptor proteins and have designed the DNA primer (SEQ ID NO: 1) coding for the amino acid sequence common (homologous) to the first membrane-spanning region and the DNA primer (SEQ ID NO: 2) which is complementary to the nucleotide sequence coding for the amino acid sequence common (homologous) to the area near the sixth membrane-spanning region. Those DNA primers have a different nucleotide sequence as compared with reported DNA primers (e.g. a set of synthetic DNA primers corresponding to the third and the sixth membrane-spanning regions (SEQ ID NO: 60 and SEQ ID NO: 61) as reported by Libert, F. et al.) and such instant primers are novel and unique.
Especially for an object of conducting an efficient elongation reaction in the PCR, the 3xe2x80x2-terminal region of the instant primers contains the nucleotide sequence which is common (homologous) among many receptor proteins. Even in other areas, the similarity (homology) at the nucleotide sequence level (base sequence level) is utilized for setting the mixed base (nucleotide) parts wherein their nucleotide sequences (base sequences) are matched for as many nucleotides (bases) as possible among many DNA for the receptor proteins. Then the present inventors have amplified cDNA derived from human brain amygdala, human pituitary gland and rat brain, found the amplified products as shown in FIG. 17 and, from those products, obtained the G protein coupled receptor protein cDNAs having the sequence as shown in FIG. 18, FIG. 19, FIG. 20, FIG. 21, FIG. 22, FIG. 23, FIG. 27, FIG. 29, FIG. 34, FIG. 37, FIG. 40, FIG. 43 or FIG. 46. Among them, the G protein coupled receptor protein cDNAs having the sequence as shown in FIG. 22, FIG. 23, FIG. 27, FIG. 29, FIG. 34, FIG. 37, FIG. 40, FIG. 43 or FIG. 46 are novel.
Further, the present inventors have selected the amino acid sequences common (homologous) to the third and the sixth membrane-spanning region each of the known G protein coupled receptor proteins and designed the DNA primers coding for the amino acid sequence common (homologous) to the third membrane-spanning region (SEQ ID NO: xcex2; SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7) and the DNA primers which are complementary to the nucleotide sequence coding for the amino acid sequence common (homologous) to the portion near the sixth membrane-spanning region (SEQ ID NO: 4, SEQ ID NO: 8 and SEQ ID NO: 9). Again, those DNA primers have different base sequences from those of the DNA primers previously reported (e.g., a set of synthetic DNA primers corresponding to the sequence of the third and the sixth membrane-spanning regions (SEQ ID NO: 60 and SEQ ID NO: 61) as reported by Libert, F. et al.) and such instant primers are novel and unique. The present inventors amplified cDNA derived from the smooth muscles of gastric pylorus of rabbits using said DNA primer and obtained G protein coupled receptor protein cDNA having the sequence of FIG. 49 or FIG. 52. Those cDNAs are novel.
Still further, the present inventors have selected the amino acid sequences common (homologous) to the second and the seventh membrane-spanning region each of the known G protein coupled receptor proteins and designed the DNA primer coding for the amino acid sequence common (homologous) to the second membrane-spanning region (SEQ ID NO: 10) and the DNA primer which is complementary to the base sequence coding for the amino acid sequence common (homologous) to the portions near the seventh membrane-spanning region (SEQ ID NO: 11). Those DNA primers have different base sequences from those of DNA primers previously reported (e.g., a set of synthetic DNA primers corresponding to the part of the third and the sixth membrane-spanning regions (SEQ ID NO: 60 and SEQ ID NO: 61) as reported by Libert, F. et al) and such instant primers are novel and unique. The present inventors amplified cDNA derived from the smooth muscles of gastric pylorus of rabbits using said DNA primer and obtained G protein coupled receptor protein cDNAs having each the sequence of FIG. 55, FIG. 56, FIG. 72, or FIG. 73. Those cDNAs are novel.
Furthermore, the present inventors have selected the amino acid sequences common (homologous) to the first and the third membrane-spanning region each of the known G protein coupled receptor proteins and designed the DNA primer coding for the amino acid sequence common (homologous) to the first membrane-spanning region (SEQ ID NO: 12) and the DNA primer which is complementary to the base sequence coding for the amino acid sequence common (homologous) to the portions near the third membrane-spanning region (SEQ ID NO: 13). Still further, the present inventors have selected the amino acid sequences common (homologous) to the third and the sixth membrane-spanning region each of the known G protein coupled receptor proteins and designed the DNA primers coding for the amino acid sequence common (homologous) to the third membrane-spanning region (SEQ ID NO: 10 and SEQ ID NO: 18) and the DNA primers which are complementary to the base sequence coding for the amino acid sequence common (homologous) to the parts near the sixth membrane-spanning region (SEQ ID NO: 15 and SEQ ID NO: 19). Further, the present inventors have selected the amino acid sequences common (homologous) to the second and the sixth membrane-spanning region each of the known G protein coupled receptor proteins and designed the DNA primer coding for the amino acid sequence common (homologous) to the second membrane-spanning region (SEQ ID NO: 16) and the DNA primer which is complementary to the base sequence coding for the amino acid sequence common (homologous) to the parts near the sixth membrane-spanning region (SEQ ID NO: 17). Those DNA primers have different base sequences from those of DNA primers previously reported (e.g., a set of synthetic DNA primers corresponding to the part of the third and the sixth membrane-spanning regions (SEQ ID NO: 60 and SEQ ID NO: 61) as reported by Libert, F. et al) and such instant primers are novel and unique.
Still another object of the present invention is to provide a G protein coupled receptor protein expressed in the pituitary gland and pancreatic xcex2 cells, a DNA comprising a DNA coding for said protein, a process for producing said protein, and use of said protein and DNA.
In order to achieve the above-mentioned aims, the present inventors have made extensive investigations. As a result, the present inventors have succeeded in amplifying cDNA derived from the human pituitary gland and the mouse pancreatic xcex2-cell strain, MIN 6, with a synthetic DNA primer for efficiently isolating G protein coupled receptor protein-encoding DNA, and have forwarded the analysis. Thus, the present inventors have succeeded in isolating novel human and mouse-derived G protein coupled receptor protein-encoding cDNAs, in determining the spartial structure thereof, and have considered that these cDNA sequences are preserved very well in the human and in the mouse, and are coding for novel receptor proteins for the same ligand. Based upon the above knowledge, the present inventors have discovered that these DNAs make it possible to obtain a cDNA having a full length open reading frame (ORF) of the receptor protein, hence, to produce the receptor protein. The inventors have further discovered that the above-mentioned receptor protein obtained when the G protein coupled receptor protein-encoding cDNA is expressed by a suitable means permits screening for a ligand to the receptor protein from the living body or from natural or non-natural compounds under guidance of data obtainable in receptor coupling tests or measurements of intracellular second messengers, etc. and further allows screening for a compound that inhibits the binding of the ligand and the receptor protein.
In one embodiment, the present inventors have carried out PCR amplification of novel human pituitary gland-derived cDNA fragments as shown in FIGS. 22 and 23, and have subcloned them to obtain a plasmid vector (p19P2). From analysis of the partial sequence, it has been clarified that the cDNA has been encoded a novel receptor protein. The synthetic DNA primers used for amplifying the cDNA are corresponding to seven hydrophobic clusters that exist in the known G protein coupled receptor proteins in common, i.e., corresponding to the first and sixth membrane-spanning regions among the membrane-spanning domains. The nucleotide sequence (SEQ ID NO: 29) has been determined from the primer region at the 5xe2x80x2 side (first membrane-spanning domain side) and has been translated into an amino acid sequence (SEQ ID NO: 24) [FIG. 22]. As a result, the second and third membrane-spanning domains have been confirmed on the hydrophobicity plotting [FIG. 58]. Similarly, the nucleotide sequence (SEQ ID NO: 30) has been determined from the primer region at the 3xe2x80x2 side (sixth membrane-spanning domain side) and has been translated into an amino acid sequence (SEQ ID NO: 25) [FIG. 23]. As a results the presence of the sixth and fifth membrane-spanning domains has been confirmed on the hydrophobicity plots [FIG. 59]. The size of the amplified cDNA is about 700 bp which is nearly comparable with the number of bases between the first membrane-spanning domain and the sixth membrane-spanning domain of the known G protein coupled receptor protein.
G protein coupled receptor proteins exert common property to some extent at an amino acid sequence level, and are forming one protein family. Therefore, data base retrieval has been carried out based upon the amino acid sequence of the subject novel receptor protein (protein encoded by cDNA included in p19P2). As a result, a high homology has been exhibited as compared with the known G protein coupled receptor protein (rat neuropeptide Y receptor protein encoded by S12863) that is shown in FIG. 60. This fact tells that the novel receptor protein of the present invention belongs to the G protein coupled receptor protein family. Moreover, the data base has been retrieved using, as a template, the amino acid sequence encoded by the DNA of the invention. It exhibits high homology to the amino acid sequences of the known G protein coupled receptor proteins, mouse-derived ligand unknown RP-23 (B40470), human-derived ligand unknown K-opioid receptor protein (P30098) and human-derived NK-2 receptor protein (JQ1059). However, none of them are in perfect agreement, from which it is learned that a novel receptor protein had been encoded. The aforementioned abbreviations in parentheses are reference numbers that are assigned when they are registered as data to NBRF-PIR/Swiss-PROT and are, usually, each called xe2x80x9cAccession Numberxe2x80x9d.
Next, by using the novel G protein coupled receptor protein-encoding cDNA fragment (p19P2) of the present invention, a cDNA having a full-length open reading frame of the receptor protein of the present invention has been obtained from human pituitary gland cDNA libraries. The nucleotide sequence analysis of a plasmid (phGR3) carrying the cDNA having a full length open reading frame of the receptor protein shows that the nucleotide sequence of a coding region of this receptor protein is represented by SEQ ID NO: 31, and the amino acid sequence deduced therefrom is represented by SEQ ID NO: 26 [FIG. 34]. Based upon the amino acid sequence, hydrophobicity plotting has been carried out. The results are shown in FIG. 36. From the hydrophobicity plotting, it has been clarified that the receptor protein of the present invention possessed seven hydrophobic domains. That is, it has been confirmed that the receptor protein encoded by the cDNA obtained according to the present invention is a seven transmembrane (membrane-spanning) G protein coupled receptor protein. An expression of mRNA for receptor genes encoded by the cDNA of the present invention has been checked by northern blotting techniques at a mRNA level, and it has been confirmed that the receptor gene has been expressed in the human pituitary gland [FIG. 35].
The present inventors have further succeeded in PCR amplification of a mouse pancreatic xcex2 cell strain, MIN6 derived cDNA fragment, and cloning of pG3-2 and pG1-10. Then, based on the nucleotide sequence of cDNA included in these two plasmid vectors, the nucleotide sequence shown in FIG. 27 has been derived. It was learned from the nucleotide sequence that the cDNA encodes a novel receptor protein. Upon translating the nucleotide sequence into an amino acid sequence, the presence of the third, fourth, fifth and sixth membrane-spanning domains has been confirmed on the hydrophobicity plots [FIG. 28]. The size of the amplified cDNA is about 400 bp which is nearly comparable with the number of bases between the third membrane-spanning domain and the sixth membrane-spanning domain of the known G protein coupled receptor protein. The amino acid sequence has been compared with amino acid sequences [FIGS. 22 and 23] encoded by the G protein coupled receptor protein cDNA included in p19P2 cloned from the human pituitary gland. As a result, homology is more than 95% [FIG. 61]. From this fact, it was estimated that the protein encoded by the cDNA included in pG3-2 is a mouse type G protein coupled receptor protein relative to the human-derived one encoded by the cDNA included in p19P2.
The present inventors have further amplified a mouse pancreatic xcex2-cell strain, MIN6-derived cDNA fragment by the PCR followed by subcloning into a plasmid vector to obtain a clone (p5S38) having a nucleotide sequence as shown in FIG. 62 . From the nucleotide sequence (SEQ ID NO: 33), it has been clarified that the cDNA encodes a novel receptor protein. Upon translating the nucleotide sequence into an amino acid sequence (SEQ ID NO: 28), the presence of the third, fourth, fifth and sixth membrane-spanning domains has been confirmed on the hydrophobicity plots [FIG. 64]. The size of the amplified DNA is about 400 bp that is nearly comparable with the known G protein coupled receptor protein. The amino acid sequence has been compared with amino acid sequences [FIGS. 22 and 23] encoded by the G protein coupled receptor protein cDNA included in p19P2 cloned from the human pituitary gland and with amino acid sequences of proteins encoded by pG3-2 and pG1-10 derived from the mouse pancreatic xcex2-cell strain. As a result, homology is more than 95% to them [FIG. 63]. This fact suggests that the protein encoded by the human-derived pituitary gland-derived p19P2, the proteins encoded by the mouse pancreatic xcex2-cell strain-derived pG3-2 and pG1-10, and the protein encoded by the mouse pancreatic xcex2-cell strain-derived p5S38, pertain to a receptor family that recognizes the same ligand.
Another object of the present invention is to provide a novel human amygdaloid nucleus-derived protein coupled receptor protein, a DNA containing a DNA coding for said G protein coupled receptor protein, a process for producing said G protein coupled receptor protein, and use of said protein and DNA.
The present inventors have synthesized DNA primers for efficiently isolating a DNA coding for G protein coupled receptor proteins, amplified an amygdaloid nucleus-derived cDNA with the above primer, and have analyzed it.
As a result, the present inventors have succeeded in isolating, from the human amygdaloid nucleus, a cDNA coding for a novel G protein coupled receptor protein and have determined its partial structure. The nucleotide sequence of the isolated cDNA is preserved very well as compared with that of the mouse glucocorticoid-induced receptor (hereinafter sometimes referred to as xe2x80x9cGIRxe2x80x9d) and is considered to be encoding a receptor protein to the same ligand (Molecular Endocrinology 5:1331-1338, 1991). It is reputed that, in the mouse, the GIR is a receptor which is induced by glucocorticoid and expressed in T-cells and is working as a receptor to immunoregulating factors in the immune system on the T-cells. The present inventors have succeeded in the isolation of this human type GIR from the human amygdaloid nucleus. Accordingly, it is suggested that the isolated GIR is expressed even in the human central nervous system to carry out some function. From these facts, it is considered that the receptor protein is strongly expressed in the human brain and in the immune system and is also functioning therein. These characterized DNAs allow one to obtain a cDNA having a full length open reading frame of the receptor and production of the receptor proteins. The receptor proteins expressed by a suitable means, furthermore, permit screening for a ligand to the receptor proteins from the living body or from natural and non-natural compounds depending on indications obtainable in receptor protein-binding experiments, measurements of intracellular second messengers, etc. It further allows one to screen for compounds capable of inhibiting the binding between the ligand and the receptor protein.
To be more specific, the present inventors have amplified, as a novel human amygdaloid nucleus-derived cDNA, one species, as shown in FIGS. 29 and 30, by PCR, cloned it, and clarified from the analysis of a partial sequence thereof that a novel receptor protein is encoded. The synthetic DNA primers used for amplifying the cDNA are corresponding to seven hydrophobic clusters that exist in the G protein coupled receptor proteins in common, i.e., corresponding to the first and sixth membrane-spanning regions among the membrane-spanning domains. The nucleotide sequence has been determined from the primer region at the 5xe2x80x2 side (first membrane-spanning domain side) and has been translated into an amino acid sequence. As a result, the second and third membrane-spanning domains have been confirmed on the hydrophobicity plotting [FIG. 31]. Similarly, the nucleotide sequence has been determined from the primer region at the 3xe2x80x2 side (sixth membrane-spanning domain side) and has been translated into an amino acid sequence. As a result, the presence of the fifth and fourth membrane-spanning domains has been confirmed on the hydrophobicity plots [FIG. 32]. The size of the amplified cDNA is about 700 bp which is nearly comparable with the number of bases of the known G protein coupled receptor protein.
The inventors have further retrieved the data base based on, as a template, the nucleotide sequence of the isolated DNA and observed high homology to the DNA that codes for mouse-derived glucocorticoid-induced receptor protein which is a widely known G protein coupled receptor protein [FIG. 33]. This result strongly suggests that the DNA of the present invention is encoding a human-type receptor protein of GIR.
Yet another object of the present invention is to provide a novel mouse pancreatic xcex2-cell strain, MIN6-derived protein coupled receptor protein, a DNA containing a DNA coding for said G protein coupled receptor protein, a process for producing said G protein coupled receptor protein, and use of said protein and DNA. The present inventors have synthesized DNA primers for efficiently isolating a DNA coding for G protein coupled receptor proteins, amplified a mouse pancreatic xcex2-cell strain, MIN6-derived cDNA with the above primer, and have analyzed it.
As a result, the present inventors have succeeded in isolating a mouse-derived cDNA coding for a novel G protein coupled receptor protein and have determined its partial structure. The isolated cDNA is homologous to known G protein coupled receptors at the nucleotide sequence level and at the amino acid sequence level and is considered to be encoding a novel receptor protein which is expressed in the mouse pancreas and is also functioning therein. These characterized DNAs allow one to obtain a cDNA having a full length open reading frame of the receptor and production of the receptor proteins. Human-derived cDNAs may be cloned by using, as a probe, said mouse-derived cDNA. The receptor proteins expressed by a suitable means, furthermore, permit screening for a ligand to the receptor protein from the living body or from natural and non-natural compounds relying on indications obtainable in receptor protein-binding experiments, measurements of intracellular second messengers, etc. It further allows one to screen for compounds capable of inhibiting the binding of the ligand with the receptor protein.
To be more specific, the present inventors have amplified, as a novel mouse pancreatic xcex2-cell strain, MIN6-derived cDNA, p3H2-17, as shown in FIGS. 37, by PCR, cloned it, and clarified from the analysis of a partial sequence thereof that a novel receptor protein is encoded. The nucleotide sequence has been translated into an amino acid sequence. As a result, the presence of the third, fourth, fifth and sixth membrane-spanning domains has been confirmed on the hydrophobicity plots [FIG. 38]. The size of the amplified cDNA is about 400 bp which is nearly comparable with that of the known G protein coupled receptor protein.
The inventors have retrieved the data base based on, as a template, the nucleotide sequence of the isolated DNA and observed 30% homology to chicken ATP receptor (P34996), 25% homology to human somatostatin receptor subtype 3 (A46226), 27% homology to human somatostatin receptor subtype 4 (JN0605), and 28% homology to bovine neuropeptide Y receptor (S28787), respectively (FIG. 39), which are known G protein coupled receptor proteins. The aforementioned abbreviations in parentheses are reference numbers that are assigned when they are registered as data to NBRF-PIR/Swiss-PROT and are, usually, each called xe2x80x9cAccession Numberxe2x80x9d.
An expression of receptor genes encoded by the cDNA fragment included in p3H2-17 of the present invention has been checked by northern blotting techniques at a mRNA level, and it has been confirmed that the receptor gene has been intensely expressed in the mouse thymus and spleen. It has been also confirmed that the receptor gene has been expressed in the mouse brain and pancreas (FIG. 65).
Next, by utilizing the information on the nucleotide sequence of the fragment included in p3H2-17, cDNA encoding a full-length open reading frame of the mouse pancreatic xcex2-cell strain, MIN6-derived G protein coupled receptor protein of the present invention has been obtained from mouse thymic and spleenic poly(A) RNA by 5xe2x80x2RACE (5xe2x80x2 rapid amplification of cDNA ends) techniques (Frohman M. A. et al., Proc. Natl. Acad. Sci. USA, 85:8998-9002 (1988); Belyavsky A. et al., Nucleic Acids Res., 17:2919-2932 (1989); Edwards J. B. D. M. et al., Nucleic Acids Res., 19:5227-5232 (1991)) and 3xe2x80x2RACE (3xe2x80x2 rapid amplification of cDNA ends) techniques (Frohman M. A. et al., Proc. Natl. Acad. Sci. USA, 85:8998-9002 (1988); Belyavsky A. et al., Nucleic Acids Res., 17:2919-2932 (1989)).
The plasmid (pMAH2-17) carrying cDNA encoding a full-length open reading frame of the receptor protein of the present invention has been subjected to sequencing analysis. As a result, the nucleotide sequence of the region coding for the receptor protein is represented by SEQ ID NO: 41 and the amino acid sequence deduced therefrom is represented by SEQ ID NO: 39 (FIG. 69). Based on the amino acid sequence, hydrophobicity plotting has been carried out. The results are shown in FIG. 70.
It has been clarified from the hydrophobicity plotting that the mouse pancreatic xcex2-cell strain, MIN6-derived receptor protein of the present invention has seven hydrophobic domains. Thus, it has been confirmed that the receptor protein encoded by the cDNA Included in pMAH2-17 according to the present invention is a seven transmembrane G protein coupled receptor protein.
Data base retrieval has been carried out based on the full-length amino acid sequence encoded by the cDNA included in pMAH2-17, and it has been observed that the amino acid sequence has 44.0% homology to mouse P2Upurinoceptor (P35383) and 38.1% homology to chicken P2Y purinoceptor (P34996), respectively (FIG. 71), which are known G protein coupled receptor proteins. The aforementioned abbreviations in parentheses are reference numbers that are assigned when they are registered as data to NBRF-PIR/SwissPROT and are, usually, each called xe2x80x9cAccession Numberxe2x80x9d. Since the receptor protein encoded by pMAH2-17 is highly homologous to prinoceptors, it is considered that there are strong possibility of a subtype within prinoceptor families. Therefore, the present inventors have carried out an electrophysiological analysis of the receptor gene in Xenopus oocytes and found significant inward currents elicited by Xenopus oocytes carrying the subject receptor gene in response to ATP stimulation (FIG. 75). As a result, it has been determined that the receptor encoded by pMAH2-17 is one of the subtypes within prinoceptor families. It has been discussed and expected that there are a variety of subtypes among purinoceptors (Pharmac. Ther., Vol. 64, pp. 445-475 (1994).
All data are supporting that the mouse pancreatic xcex2-cell strain, MIN6-derived receptor protein of the present invention (e.g., SEQ ID NO: 38 and SEQ ID NO: 39, or proteins encoded by pMAH2-17) is a novel purinoceptor subtype which is clearly distinct from chicken P2y1 purinoceptor (FEBS LETTERS, Vol. 324(2), 219-225 (1993)); mouse P2y2 or P2u purinoceptor (Proc. Natl. Acad. Sci. USA, Vol. 90, pp.5113-5117 (1993)); rat P2u or P2y2 purinoceptor (Am. J. Respir. Cell Mol. Biol., Vol. 12, pp. 27-32 (1995)); human P2u or P2y2 purinoceptor (Proc. Natl. Acad. Sci. USA, Vol. 91, pp.3275-3279 (1994)); and rat P2x purinoceptor (Nature, Vol. 371.6, pp.516-519 (1994).
It is also strongly suggested that agonists and/or antagonists related to the receptor encoded by pMAH2-17 would be useful in therapeutic or prophylactic treatment of diseases or syndromes in connection with purine ligand compounds. It is expected that the agonists of the receptor encoded by pMAH2-17 are useful as an immunomodulator or an antitumor agent, in addition they are useful in therapeutically or prophylactically treating hypertension, diabetes, cystic fibrosis, etc. It is still expected that the antagonists of the receptor encoded by pMAH2-17 are useful as hypotensive agents, analgesics, agents for therapeutically or prophylactically treating incontinence of urine, etc.
Another object of the present invention is to provide a novel human-derived protein coupled receptor protein of prinoceptor type, a DNA containing a DNA coding for said G protein coupled receptor protein, a process for producing said G protein coupled receptor protein, and use of said protein and DNA. The present inventors have synthesized DNA primers for efficiently isolating a DNA coding for prinoceptor type G protein coupled receptor proteins on the basis of the nucleotide sequence of mouse purinoceptor, amplified a human-derived cDNA with the above primer, and have analyzed it.
As a result, the present inventors have succeeded in isolating a human-derived cDNA coding for a novel G protein coupled receptor protein and have determined its full-length structure [FIG. 77]. The isolated cDNA is homologous to mouse G protein coupled receptor (purinoceptor) at the nucleotide sequence level and at the amino acid sequence level (87% homology; FIG. 79) and is considered to be encoding a novel purinoceptor protein. The receptor proteins expressed by a suitable means, furthermore, permit screening for a ligand to the receptor protein from the living body or from natural and non-natural compounds relying on indications obtainable in receptor protein-binding experiments, etc. It further allows one to screen for compounds capable of inhibiting the binding of the ligand with the receptor protein.
It is also strongly suggested that agonists and/or antagonists related to the human receptor encoded by phAH2-17 would be useful in therapeutic or prophylactic treatment of diseases or syndromes in connection with purine ligand compounds. It is expected that the agonists of the human receptor are useful as an immunomodulator or an antitumor agent, in addition they are useful in therapeutically or prophylactically treating hypertension, diabetes, cystic fibrosis, etc. It is still expected that the antagonists of the human receptor are useful as hypotensive agents, analgesics, agents for therapeutically or prophylactically treating incontinence of urine, etc.
Accordingly, one aspect of the present invention is
(1) DNAs comprising a nucleotide sequence represented by a SEQ ID NO selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 19;
(2) DNAs according to the above (1) comprising a nucleotide sequence represented by a SEQ ID NO selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 9;
(3) DNAs according to the above (1) comprising a nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2;
(4) DNAs according to the above (1) wherein the DNA is a primer for polymerase chain reaction in order to amplify a DNA coding for a G protein coupled receptor protein;
(5) a method for amplifying a DNA coding for a G protein coupled receptor protein by polymerase chain reaction techniques, which comprises:
xe2x80x83(i) carrying out a polymerase chain reaction in the presence of a mixture of
{circle around (1)} a DNA coding for G protein coupled receptor protein, said DNA being capable of acting as a template,
{circle around (2)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 1, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 3, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 5, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 6, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 7, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 10, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 14, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 16 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 18, and
{circle around (3)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 2, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 4, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 8, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 9, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 11, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 15, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 17 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 19; or
xe2x80x83(ii) carrying out a polymerase chain reaction in the presence of a mixture of
{circle around (1)} a DNA coding for G protein coupled receptor protein, said DNA being capable of acting as a template,
{circle around (2)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 1 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 12, and
{circle around (3)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 13;
(6) a method for screening a DNA library for a DNA coding for a G protein coupled receptor protein, which comprises:
xe2x80x83carrying out a polymerase chain reaction in the presence of a mixture of
{circle around (1)} said DNA library,
{circle around (2)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 1, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 3, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 5, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 6, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 7, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 10, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 14, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 16 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 18, and
{circle around (3)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 2, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 4, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 8, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 9, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 11, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 15, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 17 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 19,
to amplify selectively a template DNA coding for G protein coupled receptor protein, contained in the DNA library; or
xe2x80x83(ii) carrying out a polymerase chain reaction in the presence of a mixture of
{circle around (1)} said DNA library
{circle around (2)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ib NO: 1 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 12, and
{circle around (3)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 13, to amplify selectively a DNA coding for G protein coupled receptor protein, contained in the DNA library;
(7) a DNA coding for a G protein coupled receptor protein, which is obtained by a method according to the above (5) or (6); and
(8) G protein coupled receptor proteins encoded by a DNA according to the above (7), their peptide segments or fragments and salts thereof.
Another specific aspect of the invention is:
(9) a method for amplifying a DNA coding for G protein coupled receptor protein (e.g. a region corresponding to from the first to sixth membrane-spanning domains of G protein coupled receptor proteins or other domains thereof) by polymerase chain reaction techniques, which comprises carrying out a polymerase chain reaction in the presence of a mixture of
{circle around (1)} a DNA coding for G protein coupled receptor protein, said DNA being capable of acting as a template,
{circle around (2)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 1 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 12, and
{circle around (3)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 2, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 4, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 8, DNA primer""s comprising a nucleotide sequence represented by SEQ ID NO: 9, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 15, DNA primers comprising a nucleotide sequence represented by SEQ Ib NO: 17 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 19;
(10) a method for amplifying a DNA coding for G protein coupled receptor protein (e.g. a region corresponding to from the first to seventh membrane-spanning domains of G protein coupled receptor proteins or other domains thereof) by polymerase chain reaction techniques, which comprises carrying out a polymerase chain reaction in the presence of a mixture of
{circle around (1)} a DNA coding for G protein coupled receptor protein, said DNA being capable of acting as a template,
{circle around (2)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 1 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 12, and
{circle around (3)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 11;
(11) a method for amplifying a DNA coding for G protein coupled receptor protein (e.g. a region corresponding to from the third to sixth membrane-spanning domains of G protein coupled receptor proteins or other domains thereof) by polymerase chain reaction techniques, which comprises carrying out a polymerase chain reaction in the presence of a mixture of
{circle around (1)} a DNA coding for G protein coupled receptor protein, said DNA being capable of acting as a template,
{circle around (2)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 3, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 5, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 6, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 7, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 14 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 18, and
{circle around (3)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 2, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 4, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 8, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 9, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 15, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 17 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 19;
(12) a method for amplifying a DNA coding for G protein coupled receptor protein (e.g. a region corresponding to from the third to seventh membrane-spanning domains of G protein coupled receptor proteins or other domains thereof) by polymerase chain reaction techniques, which comprises carrying out a polymerase chain reaction in the presence of a mixture of
{circle around (1)} a DNA coding for G protein coupled receptor protein, said DNA being capable of acting as a template,
{circle around (2)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 3, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 5, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 6, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 7, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 14 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 18, and
{circle around (3)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 11;
(13) a method for amplifying a DNA coding for G protein coupled receptor protein (e.g. a region corresponding to from the second to sixth membrane-spanning domains of G protein coupled receptor proteins or other domains thereof) by polymerase chain reaction techniques, which comprises carrying out a polymerase chain reaction in the presence of a mixture of
{circle around (1)} a DNA coding for G protein coupled receptor protein, said DNA being capable of acting as a template,
{circle around (2)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 10 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 16, and
{circle around (3)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 2, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 4, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 8, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 9, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 15, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 17 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 19;
(14) a method for amplifying a DNA coding for G protein coupled receptor protein (e.g. a region corresponding to from the second to seventh membrane-spanning domains of G protein coupled receptor proteins or other domains thereof) by polymerase chain reaction techniques, which comprises carrying out a polymerase chain reaction in the presence of a mixture of
{circle around (1)} a DNA coding for G protein coupled receptor protein, said DNA being capable of acting as a template,
{circle around (2)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 10 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 16, and
{circle around (3)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 11;
(15) a method-for amplifying a DNA coding for G protein coupled receptor protein (e.g., a region corresponding to from the first to third membrane-spanning domains of G protein coupled receptor proteins or other domains thereof) by polymerase chain reaction techniques, which comprises carrying out a polymerase chain reaction in the presence of a mixture of
{circle around (1)} a DNA coding for G protein coupled receptor protein, said DNA being capable of acting as a template,
{circle around (2)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 1 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 12, and
{circle around (3)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 13;
(16) a method for amplifying a DNA coding for G protein coupled receptor protein by polymerase chain reaction techniques, which comprises carrying out a polymerase chain reaction in the presence of a mixture of
{circle around (1)} a DNA coding for G protein coupled receptor protein, said DNA being capable of acting as a template,
{circle around (2)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 1, and
{circle around (3)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 2;
(17) a method for amplifying a DNA coding for G protein coupled receptor protein by polymerase chain reaction techniques, which comprises carrying out a polymerase chain reaction in the presence of a mixture of
{circle around (1)} a DNA coding for G protein coupled receptor protein, said DNA being capable of acting as a template,
{circle around (2)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 3, and
{circle around (3)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 4;
(18) a method for amplifying a DNA coding for G protein coupled receptor protein by polymerase chain reaction techniques, which comprises carrying out a polymerase chain reaction in the presence of a mixture of
{circle around (1)} a DNA coding for G protein coupled receptor protein, said DNA being capable of acting as a template,
{circle around (2)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 6, and
{circle around (3)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 8;
(19) a method for amplifying a DNA coding for G protein coupled receptor protein by polymerase chain reaction techniques, which comprises carrying out a polymerase chain reaction in the presence of a mixture of
{circle around (1)} a DNA coding for G protein coupled receptor protein, said DNA being capable of acting as a template,
{circle around (2)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 10, and
{circle around (3)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 11;
(20) a method for amplifying DNA coding for a G protein coupled receptor protein which comprises
xe2x80x83(i) carrying out a polymerase chain reaction in the presence of a mixture of
{circle around (1)} a DNA coding for G protein coupled receptor protein, said DNA being capable of acting as a template,
{circle around (2)} at least one DNA primer which is capable of binding with the 3xe2x80x2-side nucleotide sequence of the xe2x88x92 chain (minus chain) of the template DNA coding for G protein coupled receptor protein to allow the extension of the + chain (plus chain) in the 5xe2x80x2xe2x86x923xe2x80x2 direction, said DNA primer being selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 1, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 3, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 5, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 6, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 7, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 10, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 12, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 14, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 16 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 18, and
{circle around (3)} at least one DNA primer which is capable of binding with the 3xe2x80x2-side nucleotide sequence of the + chain (plus chain) of the template DNA coding for G protein coupled receptor protein to allow the extension of the xe2x88x92 chain (minus chain) in the 5xe2x80x2xe2x86x923xe2x80x2 direction, said DNA primer being selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 2, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 4, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 8, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 9, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 11, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 15, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 17 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 19, or
xe2x80x83(ii) carrying out a polymerase chain reaction in the presence of a mixture of
{circle around (1)} a DNA coding for G protein coupled receptor protein, said DNA being capable of acting as a template,
{circle around (2)} at least one DNA primer which is capable of binding with the 3xe2x80x2-side nucleotide sequence of the xe2x88x92 chain (minus chain) of the template DNA coding for G protein coupled receptor protein to allow the extension of the + chain (plus chain) in the 5xe2x80x2xe2x86x923xe2x80x2 direction, said DNA primer being selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 1 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 12, and
{circle around (3)} at least one DNA primer which is capable of binding with the 3xe2x80x2-side nucleotide sequence of the + chain (plus chain) of the template DNA coding for G protein coupled receptor protein to allow the extension of the xe2x88x92 chain (minus chain) in the 5xe2x80x2xe2x86x923xe2x80x2 direction, said DNA primer being selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 13;
(21) a method for screening DNA libraries for a DNA coding for G protein coupled receptor protein (e.g. from the first to sixth membrane-spanning domains or other domains of G protein coupled receptor protein), which comprises carrying out a polymerase chain reaction in the presence of a mixture of
{circle around (1)} said DNA library,
{circle around (2)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 1 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 12, and
{circle around (3)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 2, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 4, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 8, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 9, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 15, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 17 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 19,
to amplify selectively a template DNA coding for G protein coupled receptor protein (e.g. from the first to sixth membrane-spanning domains or other domains of G protein coupled receptor protein), contained in the DNA library;
(22) a method for screening DNA libraries for a DNA coding for G protein coupled receptor protein (e.g. from the first to seventh membrane-spanning domains or other domains of G protein coupled receptor protein), which comprises carrying out a polymerase chain reaction in the presence of a mixture of
{circle around (1)} said DNA library,
{circle around (2)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 1 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 12, and
{circle around (3)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 11,
to amplify selectively a template DNA coding for G protein coupled receptor protein (e.g. from the first to seventh membrane-spanning domains or other domains of G protein coupled receptor protein), contained in the DNA library;
(23) a method for screening DNA libraries for a DNA coding for G protein coupled receptor protein (e.g. from the third to sixth membrane-spanning domains or other domains of G protein coupled receptor protein), which comprises carrying out a polymerase chain reaction in the presence of a mixture of
{circle around (1)} said DNA library,
{circle around (2)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 3, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 5, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 6, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 7, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 14 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 18, and
{circle around (3)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 2, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 4, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 8, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 9, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 15, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 17 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 19,
to amplify selectively a template DNA coding for G protein coupled receptor protein (e.g. from the third to sixth membrane-spanning domains or other domains of G protein coupled receptor protein), contained in the DNA library;
(24) a method for screening DNA libraries for a DNA coding for G protein coupled receptor protein (e.g. from the third to seventh membrane-spanning domains or other domains of G protein coupled receptor protein), which comprises carrying out a polymerase chain reaction in the presence of a mixture of
{circle around (1)} said DNA library,
{circle around (2)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 3, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 5, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 6, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 7, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 14 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 18, and
{circle around (3)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 11,
to amplify selectively a template DNA coding for G protein coupled receptor protein (e.g. from the third to seventh membrane-spanning domains or other domains of G protein coupled receptor protein), contained in the DNA library;
(25) a method for screening DNA libraries for a DNA coding for G protein coupled receptor protein (e.g. from the second to sixth membrane-spanning domains or other domains of G protein coupled receptor protein), which comprises carrying out a polymerase chain reaction in the presence of a mixture of
{circle around (1)} said DNA library,
{circle around (2)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 10 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 16, and
{circle around (3)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 2, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 4, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 8, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 9, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 15, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 17 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 19,
to amplify selectively a template DNA coding for G protein coupled receptor protein (e.g. from the second to sixth membrane-spanning domains or other domains of G protein coupled receptor protein), contained in the DNA library;
(26) a method for screening DNA libraries for a DNA coding for G protein coupled receptor protein (e.g. from the second to seventh membrane-spanning domains or other domains of G protein coupled receptor protein), which comprises carrying out a polymerase chain reaction in the presence of a mixture of
{circle around (1)} said DNA library,
{circle around (2)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 10 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 16, and
{circle around (3)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 11,
to amplify selectively a template DNA coding for G protein coupled receptor protein (e.g. from the second to seventh membrane-spanning domains or other domains of G protein coupled receptor protein), contained in the DNA library;
(27) a method for screening DNA libraries for a DNA coding for G protein coupled receptor protein (e.g. from the first to third membrane-spanning domains or other domains of G protein coupled receptor protein), which comprises carrying out a polymerase chain reaction in the presence of a mixture of
{circle around (1)} said DNA library,
{circle around (2)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 1 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 12, and
{circle around (3)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 13,
to amplify selectively a template DNA coding for G protein coupled receptor protein (e.g. from the first to third membrane-spanning domains or other domains of G protein coupled receptor protein), contained in the DNA library;
(28) a method for screening DNA libraries for a DNA coding for G protein coupled receptor protein, which comprises carrying out a polymerase chain reaction in the presence of a mixture of
{circle around (1)} said DNA library,
{circle around (2)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 1, and
{circle around (3)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 2,
to amplify selectively the template DNA coding for G protein coupled receptor protein, contained in the DNA library;
(29) a method for screening DNA libraries to detect a DNA coding for G protein coupled receptor protein, which comprises carrying out a polymerase chain reaction in the presence of a mixture of
{circle around (1)} said DNA library,
{circle around (2)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 3, and
{circle around (3)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 4,
to amplify selectively a template DNA coding for G protein coupled receptor protein, contained in the DNA library;
(30) a method for screening DNA libraries for a DNA coding for G protein coupled receptor protein, which comprises carrying out a polymerase chain reaction in the presence of a mixture of
{circle around (1)} said DNA library,
{circle around (2)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 6, and
{circle around (3)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 8,
to amplify selectively a template DNA coding for G protein coupled receptor protein, contained in the DNA library;
(31) a method for screening DNA libraries for a DNA coding for G protein coupled receptor protein, which comprises carrying out a polymerase chain reaction in the presence of a mixture of
{circle around (1)} said DNA library,
{circle around (2)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 10, and
{circle around (3)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 11,
to amplify selectively a template DNA coding for G protein coupled receptor protein, contained in the DNA library; and
(32) a method for screening DNA libraries according to any of the above (6), and (21) to (31) wherein said DNA library is derived from an origin selected from the group consisting of human tissues and human cells. Examples of such human tissues include adrenal, umbilical cord, brain, tongue, liver, lymph gland, lung, thymus, placenta, peritoneum, retina, spleen, heart, smooth muscle, intestine, vessel, bone, kidney, skin, fetus, mammary gland, ovary, testis, pituitary gland, pancreas, submandibular gland, spine, prostate gland, stomach, thyroid gland, trachea (windpipe), skeletal muscle, uterus, adipose tissue, urinary bladder, cornea, olfactory bulb, bone marrow, amnion, etc. Examples of such human cells include nerve cells, epithelial cells, endothelial cells, leukocytes, lymphocytes, gliacytes, fibroblasts, keratinized cells, osteoblasts, osteoclasts, astrocytes, melanocytes, various carcinomas, various sarcomas, various cells derived from the above-mentioned human tissues.
Yet another aspect of the present invention is a degenerate deoxynucleotide which has an oligonucleotide sequence to which a SEQ ID NO selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 19 is assigned.
Another aspect of the present invention is
(33) a G protein coupled receptor protein comprising an amino acid sequence selected from the group consisting of amino acid sequences represented by SEQ ID NO: 24 and/or SEQ ID NO: 25 and substantial equivalents to the amino acid sequence represented by SEQ ID NO: 24 or SEQ ID NO: 25; or a salt thereof;
(34) a G protein coupled receptor protein according to the above (33) comprising an amino acid sequence selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 26 and substantial equivalents to the amino acid sequence represented by SEQ ID NO: 26; or a salt thereof;
(35) a G protein coupled receptor protein comprising an amino acid sequence selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 27 and substantial equivalents to the amino acid sequence represented by SEQ ID NO: 27; or a salt thereof;
(36) a G protein coupled receptor protein comprising an amino acid sequence selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 28 and substantial equivalents to the amino acid sequence represented by SEQ ID NO: 28; or a salt thereof;
(37) a G protein coupled receptor protein comprising an amino acid sequence selected from the group consisting of amino acid sequences represented by SEQ ID NO: 34 and/or SEQ ID NO: 35 and substantial equivalents to the amino acid sequence represented by SEQ ID NO: 34 or SEQ ID NO: 35; or a salt thereof;
(38) a G protein coupled receptor protein comprising an amino acid sequence selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 38 and substantial equivalents to the amino acid sequence represented by SEQ ID NO: 38; or a salt thereof;
(39) a G protein coupled receptor protein according to the above (38) comprising an amino acid sequence selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 39 and substantial equivalents to the amino acid sequence represented by SEQ ID NO: 39; or a salt thereof;
(40) a G protein coupled receptor protein comprising an amino acid sequence represented by SEQ ID NO: 56 and substantial equivalents to the amino acid sequence represented by SEQ ID NO: 56; or a salt thereof;
(41) a peptide segment or fragment of a G protein coupled receptor protein according to any of the above (33) to (40), a modified derivative thereof or a salt thereof;
(42) a DNA which comprises a nucleotide sequence coding for a G protein coupled receptor protein of the above (33);
(43) a DNA which comprises a nucleotide sequence coding for a G protein coupled receptor protein of the above (34);
(44) a DNA which comprises a nucleotide sequence coding for a G protein coupled receptor protein of the above (35);
(45) a DNA which comprises a nucleotide sequence coding for a G protein coupled receptor protein of the above (36);
(46) a DNA which comprises a nucleotide sequence coding for a G protein coupled receptor protein of the above (37);
(47) a DNA which comprises a nucleotide sequence coding for a G protein coupled receptor protein of the above (38);
(48) a DNA which comprises a nucleotide sequence coding for a G protein coupled receptor protein of the above (39);
(49) a DNA which comprises a nucleotide sequence coding for a G protein coupled receptor protein of the above (40);
(50) a DNA of the above (42) comprising a nucleotide sequence represented by SEQ ID NO: 29 and/or SEQ ID NO: 30;
(51) a DNA of the above (43) comprising a nucleotide sequence represented by SEQ ID NO: 31;
(52) a DNA of the above (44) comprising a nucleotide sequence represented by SEQ ID NO: 32;
(53) a DNA of the above (45) comprising a nucleotide sequence represented by SEQ ID NO: 33;
(54) a DNA of the above (46) comprising a nucleotide sequence represented by SEQ ID NO: 36 and/or SEQ ID NO: 37;
(55) a DNA of the above (47) comprising a nucleotide sequence represented by SEQ ID NO: 40;
(56) a DNA of the above (48) comprising a nucleotide sequence represented by SEQ ID NO: 41;
(57) a DNA of the above (49) comprising a nucleotide sequence represented by SEQ ID NO: 57;
(58) a vector comprising a DNA according to any of the above (42) to (57);
(59) a transformant (including a transfectant) carrying a vector of the above (58);
(60) a process for producing a G protein coupled receptor protein or a salt thereof according to any of the above (33) to (40), which comprises culturing a transformant of the above (59) to express said G protein coupled receptor protein on the membrane of the transformant;
(61) a method for determining a ligand to a G protein coupled receptor protein according to any of the above (33) to (40), which comprises contacting
(i) at least one component selected from the group consisting of G protein coupled receptor proteins or salts thereof according to any of the above (33) to (40), peptide fragments or segments or salts thereof according to the above (41), and mixtures thereof, with
(ii) at least one compound to be tested;
(62) a screening method for a compound capable of inhibiting the binding of a G protein coupled receptor protein according to any of the above (33) to (40) with a ligand, which comprises carrying out a comparison between:
(i) at least one case where said ligand is contacted with at least one component selected from the group consisting of G protein coupled receptor proteins or salts thereof according to any of the above (33) to (40), peptide fragments or segments or salts thereof according to the above (41), and mixtures thereof, and
(ii) at least one case where said ligand together with a compound to be tested is contacted with at least one component selected from the group consisting of G protein coupled receptor proteins or salts thereof according to any of the above (33) to (40), peptide fragments or segments or salts thereof according to the above (41), and mixtures thereof;
(63) a kit for the screening of one or more compounds capable of inhibiting the binding of a G protein coupled receptor protein according to any of the above (33) to (40), with a ligand, which comprises at least one component selected from the group consisting of G protein coupled receptor proteins or salts thereof according to any of the above (33) to (40), peptide fragments or segments or salts thereof according to the above (41), and mixtures thereof; and
(64) an antibody against at least one component selected from the group consisting of G protein coupled receptor proteins or salts thereof according to any of the above (33) to (40), peptide fragments or segments or salts thereof according to the above (41), and mixtures thereof.
Yet another aspect of the present invention is
(65) a G protein coupled receptor protein according to the above (33) comprising
(i) an amino acid sequence selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 24, amino acid sequences wherein one or more amino acid residues (preferably from 2 to 30 amino acid residues, more preferably from 2 to 10 amino acid residues) are deleted from the amino acid sequence of SEQ ID NO: 24, amino acid sequences wherein one or more amino acid residues (preferably from 2 to 30 amino acid residues, more preferably from 2 to 10 amino acid residues) are added to the amino acid sequence of SEQ ID NO: 24, and amino acid sequences wherein one or more amino acid residues (preferably from 2 to 30 amino acid residues, more preferably from 2 to 10 amino acid residues) in the amino acid sequence of SEQ ID NO: 24 are substituted with one or more other amino acid residues, or/and
(ii) an amino acid sequence selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 25, amino acid sequences wherein one or more amino acid residues (preferably from 2 to 30 amino acid residues, more preferably from 2 to 10 amino acid residues) are deleted from the amino acid sequence of SEQ ID NO: 25, amino acid sequences wherein one or more amino acid residues (preferably from 2 to 30 amino acid residues, more preferably from 2 to 10 amino acid residues) are added to the amino acid sequence of SEQ ID NO: 25, and amino acid sequences wherein one or more amino acid residues (preferably from 2 to 30 amino acid residues, more preferably from 2 to 10 amino acid residues) in the amino acid sequence of SEQ ID NO: 25 are substituted with one or more other amino acid residues,
or a salt thereof;
(66) a G protein coupled receptor protein according to the above (34) comprising an amino acid sequence selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 26, amino acid sequences wherein one or more amino acid residues (preferably from 2 to 30 amino acid residues, more preferably from 2 to 10 amino acid residues) are deleted from the amino acid sequence of SEQ ID NO: 26, amino acid sequences wherein one or more amino acid residues (preferably from 2 to 30 amino acid residues, more preferably from 2 to 10 amino acid residues) are added to the amino acid sequence of SEQ ID NO: 26, and amino acid sequences wherein one or more amino acid residues (preferably from 2 to 30 amino acid residues, more preferably from 2 to 10 amino acid residues) in the amino acid sequence of SEQ ID NO: 26 are substituted with one or more other amino acid residues, or a salt thereof;
(67) a G protein coupled receptor protein according to the above (35) comprising an amino acid sequence selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 27, amino acid sequences wherein one or more amino acid residues (preferably from 2 to 30 amino acid residues, more preferably from 2 to 10 amino acid residues) are deleted from the amino acid sequence of SEQ ID NO: 27, amino acid sequences wherein one or more amino acid residues (preferably from 2 to 30 amino acid residues, more preferably from 2 to 10 amino acid residues) are added to the amino acid sequence of SEQ ID NO: 27, and amino acid sequences wherein one or more amino acid residues (preferably from 2 to 30 amino acid residues, more preferably from 2 to 10 amino acid residues) in the amino acid sequence of SEQ ID NO: 27 are substituted with one or more other amino acid residues, or a salt thereof;
(68) a G protein coupled receptor protein according to the above (36) comprising an amino acid sequence selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 28, amino acid sequences wherein one or more amino acid residues (preferably from 2 to 30 amino acid residues, more preferably from 2 to 10 amino acid residues) are deleted from the amino acid sequence of SEQ ID NO: 28, amino acid sequences wherein one or more amino acid residues (preferably from 2 to 30 amino acid residues, more preferably from 2 to 10 amino acid residues) are added to the amino acid sequence of SEQ ID NO: 28, and amino acid sequences wherein one or more amino acid residues (preferably from 2 to 30 amino acid residues, more preferably from 2 to 10 amino acid residues) in the amino acid sequence of SEQ ID NO: 28 are substituted with one or more other amino acid residues, or a salt thereof;
(69) a G protein coupled receptor protein according to the above (37) comprising
(i) an amino acid sequence selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 34, amino acid sequences wherein one or more amino acid residues (preferably from 2 to 30 amino acid residues, more preferably from 2 to 10 amino acid residues) are deleted from the amino acid sequence of SEQ ID NO: 34, amino acid sequences wherein one or more amino acid residues (preferably from 2 to 30 amino acid residues, more preferably from 2 to 10 amino acid residues) are added to the amino acid sequence of SEQ ID NO: 34, and amino acid sequences wherein one or more amino acid residues (preferably from 2 to 30 amino acid residues, more preferably from 2 to 10 amino acid residues) in the amino acid sequence of SEQ ID NO: 34 are substituted with one or more other amino acid residues, or/and
(ii) an amino acid sequence selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 35, amino acid sequences wherein one or more amino acid residues (preferably from 2 to 30 amino acid residues, more preferably from 2 to 10 amino acid residues) are deleted from the amino acid sequence of SEQ ID NO: 35, amino acid sequences wherein one or more amino acid residues (preferably from 2 to 30 amino acid residues, more preferably from 2 to 10 amino acid residues) are added to the amino acid sequence of SEQ ID NO: 35, and amino acid sequences wherein one or more amino acid residues (preferably from 2 to 30 amino acid residues, more preferably from 2 to 10 amino acid residues) in the amino acid sequence of SEQ ID NO: 35 are substituted with one or more other amino acid residues,
or a salt thereof;
(70) a G protein coupled receptor protein according to the above (38) comprising an amino acid sequence selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 38, amino acid sequences wherein one or more amino acid residues (preferably from 2 to 30 amino acid residues, more preferably from 2 to 10 amino acid residues) are deleted from the amino acid sequence of SEQ ID NO: 38, amino acid sequences wherein one or more amino acid residues (preferably from 2 to 30 amino acid residues, more preferably from 2 to 10 amino acid residues) are added to the amino acid sequence of SEQ ID NO: 38, and amino acid sequences wherein one or more amino acid residues (preferably from 2 to 30 amino acid residues, more preferably from 2 to 10 amino acid residues) in the amino acid sequence of SEQ ID NO: 38 are substituted with one or more other amino acid residues, or a salt thereof;
(71) a G protein coupled receptor protein according to the above (39) comprising an amino acid sequence selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 39, amino acid sequences wherein one or more amino acid residues (preferably from 2 to 30 amino acid residues, more preferably from 2 to 10 amino acid residues) are deleted from the amino acid sequence of SEQ ID NO: 39, amino acid sequences wherein one or more amino acid residues (preferably from 2 to 30 amino acid residues, more preferably from 2 to 10 amino acid residues) are added to the amino acid sequence of SEQ ID NO: 39, and amino acid sequences wherein one or more amino acid residues (preferably from 2 to 30 amino acid residues, more preferably from 2 to 10 amino acid residues) in the amino acid sequence of SEQ ID NO: 39 are substituted with one or more other amino acid residues, or a salt thereof;
(72) a G protein coupled receptor protein according to the above (40) comprising an amino acid sequence selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 56, amino acid sequences wherein one or more amino acid residues (preferably from 2 to 30 amino acid residues, more preferably from 2 to 10 amino acid residues) are deleted from the amino acid sequence of SEQ ID NO: 56, amino acid sequences wherein one or more amino acid residues (preferably from 2 to 30 amino acid residues, more preferably from 2 to 10 amino acid residues) are added to the amino acid sequence of SEQ ID NO: 56, and amino acid sequences wherein one or more amino acid residues (preferably from 2 to 30 amino acid residues, more preferably from 2 to 10 amino acid residues) in the amino acid sequence of SEQ ID NO: 56 are substituted with one or more other amino acid residues, or a salt thereof;
(73) a method for determining a ligand according to the above (61) wherein said ligand is selected from the group consisting of angiotensin, bombesin, canavinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine, vasopressin, oxytocin, VIP (vasoactive intestinal and related peptides), somatostatin, dopamine, motilin, amylin, bradykinin, CGRP (calcitonin gene related peptides), adrenomedullin, leukotriene, pancreastatin, prostaglandin, thromboxanes, adenosine, adrenaline, xcex1- and xcex2-chemokine (IL-8, GROxcex1, GROxcex2, GROxcex3, NAP-2, ENA-78, PF4, IP10, GCP-2, MCP-1, HC14, MCP-3, I-309, MIP1xcex1, MIP-1xcex2, RANTES, etc.), endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptides and galanin;
(74) a method for the screening of a compound or a salt thereof capable of inhibiting the binding of a ligand with a G protein coupled receptor protein according to any of the above (33) to (40), which comprises measuring amounts of a labeled ligand bound to the said G protein coupled receptor protein in at least two cases:
(i) where the labeled ligand is contacted with at least one component selected from the group consisting of G protein coupled receptor proteins or salts thereof according to any of the above (33) to (40), peptide fragments or segments or salts thereof according to the above (41), and mixtures thereof, and
(ii) where the labeled ligand together with a compound to be tested is contacted with at least one component elected from the group consisting of G protein coupled receptor proteins or salts thereof according to any of the above (33) to (40), peptide fragments or segments or salts thereof according to the above (41), and mixtures thereof,
and comparing the measured amounts of the labeled ligand;
(75) a method for the screening of a compound or a salt thereof capable of inhibiting the binding of a ligand with a G protein coupled receptor protein according to any of the above (33) to (40), which comprises measuring amounts of a labeled ligand bound to a cell comprising the said G protein coupled receptor protein in at least two cases:
(i) where the labeled ligand is contacted with the said cell, and
(ii) where the labeled ligand together with a compound to be tested is contacted with the said cell,
and comparing the measured amounts of the labeled ligand;
(76) a method for the screening of a compound or a salt thereof capable of inhibiting the binding of a ligand with a G protein coupled receptor protein according to any of the above (33) to (40), which comprises measuring amounts of a labeled ligand bound to a membrane fraction of a cell comprising the said G protein coupled receptor protein in at least two cases:
(i) where the labeled ligand is contacted with the said membrane fraction, and
(ii) where the labeled ligand together with a compound to be tested is contacted with the membrane fraction,
and comparing the measured amounts of the labeled ligand;
(77) a method for the screening of a compound or a salt thereof capable of inhibiting the binding of a ligand with a G protein coupled receptor protein according to any of the above (33) to (40), which comprises measuring amounts of a labeled ligand bound to said G protein coupled receptor protein in at least two cases:
(i) where the labeled ligand is contacted with a G protein coupled receptor protein according to any of the above (33) to (40) which is expressed on the membrane of a transformant according to the above (59) during incubation of the transformant, and
(ii) where the labeled ligand together with a compound to be tested is contacted with the G protein coupled receptor protein according to any of the above (33) to (40) which is expressed on the membrane of a transformant according to the above (59) during incubation of the transformant, and comparing the measured amounts of the labeled ligand;
(78) a method for the screening of a compound or a salt thereof capable of inhibiting the binding of a ligand with a G protein coupled receptor protein according to any of the above (33) to (40), which comprises measuring G protein coupled receptor protein-mediated cell-stimulating activities in at least two cases:
(i) where a compound capable of activating the G protein coupled receptor protein according to any of the above (33) to (40) is contacted with a cell comprising the said G protein coupled receptor protein, and
(ii) where the compound capable of activating the G protein together with a compound to be tested is contacted with the cell comprising the said G protein coupled receptor protein,
and comparing the measured cell-stimulating activities;
(79) a method for the screening of a compound or a salt thereof capable of inhibiting the binding of a ligand with a G protein coupled receptor protein according to any of the above (33) to (40), which comprises measuring G protein coupled receptor protein-mediated cell-stimulating activities in at least two cases:
(i) where a compound capable of activating the G protein coupled receptor protein according to any of the above (33) to (40) is contacted with a G protein coupled receptor protein according to any of the above (33) to (40) which is expressed on the membrane of a transformant according to the above (59) during incubation of the transformant, and
(ii) where the compound capable of activating the G protein together with a compound to be tested is contacted with the G protein coupled receptor protein according to any of the above (33) to (40) which is expressed on the membrane of a transformant according to the above (59) during incubation of the transformant,
and comparing the measured cell-stimulating activities;
(80) a method according to the above (78) or (79) wherein said compound capable of activating the G protein coupled receptor protein according to any of the above (33) to (40) is selected from the group consisting of angiotensin, bombesin, canavinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine, vasopressin, oxytocin, VIP (vasoactive intestinal and related peptides), somatostatin, dopamine, motilin, amylin, bradykinin, CGRP (calcitonin gene related peptides), adrenomedullin, leukotriene, pancreastatin, prostaglandin, thromboxane, adenosine, adrenaline, xcex1- and xcex2-chemokine (IL-8, GROxcex1, GROxcex2, GROxcex3, NAP-2, ENA-78, PF4, IP10, GCP-2, MCP-1, HC14, MCP-3, I-309, MIP1xcex1, MIP-1xcex2, RANTES, etc.), endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptides and galanin;
(81) a compound which is determined through a method according to any of the above (62) and (74) to (80) or a salt thereof;
(82) a pharmaceutical composition comprising an effective amount of a compound according to the above (81) or a salt thereof;
(83) a screening kit according to the above (63), comprising a cell comprising a G protein coupled receptor protein according to any of the above (33) to (40);
(84) a screening kit according to the above (63), comprising a membrane fraction derived from a cell comprising a G protein coupled receptor protein according to any of the above (33) to (40);
(85) a screening kit according to the above (63), comprising a cell of the (59) or (109) mentioned herein below;
(86) a screening kit according to the above (63), comprising a membrane fraction derived from a cell of the (59) or (109);
(87) a compound which is determined by means of a screening kit according to any of the above (63) and (83) to (86) or a salt thereof;
(88) a pharmaceutical composition comprising an effective amount of a compound according to the above (87) or a salt thereof; and
(89) a method for measuring at least one component selected from the group consisting of G protein coupled receptor proteins or salts thereof according to any of the above (33) to (40), peptide fragments or segments or salts thereof according to the above (41), and mixtures thereof, which comprises contacting an antibody according to the above (64) with the component selected from the group consisting of G protein coupled receptor proteins or salts thereof according to any of the above (33) to (40), peptide segments or salts thereof according to the above (41), and mixtures thereof.
Still another aspect of the present invention is
(90) a ligand to a G protein coupled receptor protein according to any of the above (33) to (40), which is determined through the following step of:
contacting (i) at least one component selected from the group consisting of G protein coupled receptor proteins or salts thereof according to any of the above (33) to (40), peptide fragments or segments or salts thereof according to the above (41), and mixtures thereof,
with (ii) at least one compound to be examined; and
(91) a compound capable of inhibiting the binding of a G protein coupled receptor protein according to any of the above (33) to (40) with a ligand, which is determined through carrying out a comparison between:
(i) at least one case where said ligand is contacted with at least one component selected from the group consisting of G protein coupled receptor proteins or salts thereof according to any of the above (33) to (40), peptide fragments or segments or salts thereof according to the above (41), and mixtures thereof, and
(ii) at least one case where said ligand together with a compound to be tested is contacted with at least one component selected from the group consisting of G protein coupled receptor proteins or salts thereof according to any of the above (33) to (40), peptide fragments or segments or salts thereof according to the above (41), and mixtures thereof.
Another aspect of the present invention is
(92) a recombinant G protein coupled receptor protein and a salt thereof which is obtained by the expression of a DNA according to any of the above (42) to (57), or a modified or fragmented derivative thereof;
(93) a method for amplifying a DNA coding for G protein coupled receptor protein by polymerase chain reaction techniques, which comprises carrying out a polymerase chain reaction in the presence of a mixture of
(1) a DNA coding for G protein coupled receptor protein, said DNA being capable of acting as a template, and
(2) at least one DNA primer selected from the group consisting of DNA primers comprising either SEQ ID NO: 1 or SEQ ID NO: 2; and
(94) a method for screening DNA libraries for a DNA coding for G protein coupled receptor protein, which comprises carrying out a polymerase chain reaction in the presence of a mixture of
(1) said DNA library, and
(2) at least one DNA primer selected from the group consisting of DNA primers comprising either SEQ ID NO: 1 or SEQ ID NO: 2,
to amplify selectively the DNA coding for G protein coupled receptor protein, contained in the DNA library.
Yet another aspect of the present invention is
(95) a monoclonal antibody against at least one component selected from the group consisting of G protein coupled receptor proteins or salts thereof according to any of the above (33) to (40), peptide fragments or segments or salts thereof according to the above (41), and mixtures thereof;
(96) a preparation of purified polyclonal antibodies against at least one component selected from the group consisting of G protein coupled receptor proteins or salts thereof according to any of the above (33) to (40), peptide fragments or segments or salts thereof according to the above (41), and mixtures thereof;
(97) an immunoassay for detecting a G protein coupled receptor protein which comprising
(i) incubating a sample to be tested with an antibody according to the above (64) to allow formation of an antigen-antibody complex; and
(ii) detecting an antigen-antibody complex formed in step (i); and
(98) an immunoassay for detecting antibodies against a G protein coupled receptor protein which comprising
(i) incubating a sample to be tested with at least one component selected from the group consisting of G protein coupled receptor proteins or salts thereof according to any of the above (33) to (40), peptide fragments or segments or salts thereof according to the above (41), and mixtures thereof to allow formation of an antigen-antibody complex; and
(ii) detecting an antigen-antibody complex formed in step (a).
Still another aspect of the present invention is
(99) an antisense DNA or RNA which comprises a nucleotide sequence complementary to at least a portion of a DNA according to any of the above (42) to (57), said antisense DNA or RNA being hybridizable to said DNA according to any of the above (42) to (57);
(100) an antisense DNA or RNA according to the above (99) wherein said antisense DNA or RNA comprises the 5xe2x80x2 end hairpin loop, 5xe2x80x2 end 6-base-pair repeat, 5xe2x80x2 end untranslated region, protein translation initiation site or codon, ORF translation initiation site or codon, 3xe2x80x2-untranslated region, 3xe2x80x2 end palindrome region, or 3xe2x80x2 end hairpin loop of a G protein coupled receptor protein DNA according to any of the above (42) to (57);
(101) an antisense DNA or RNA according to the above (99) in a pharmaceutically acceptable carrier;
(102) an antisense DNA or RNA according to the above (99) comprising from 2 to 50 nucleotides;
(103) a method for modulating the activity of a G protein coupled receptor protein comprising contacting cells expressing the G protein coupled receptor protein with an antisense DNA or RNA according to the above (99);
(104) a method for producing an antibody against a G protein coupled receptor protein according to any of the above (33) to (40), which comprises administering to an individual at least one component selected from the group consisting of G protein coupled receptor proteins or salts thereof according to any of the above (33) to (40), peptide fragments or segments or salts thereof according to the above (41), and mixtures thereof; and
(105) a method for producing a hybridoma which produces a monoclonal antibody against a G protein coupled receptor protein according to any of the above (33) to (40), which comprises
(i) immunizing an individual with at least one component selected from the group consisting of G protein coupled receptor proteins or salts thereof according to any of the above (33) to (40), peptide fragments or segments or salts thereof according to the above (41), and mixtures thereof;
(ii) immortalizing antibody producing cells from the immunized individual;
(iii) selecting an immortal cell which produces antibodies reactive with the G protein coupled receptor protein; and
(iv) growing said immortal cell.
Yet another aspect of the present invention is
(106) a PCR screening kit for a DNA (or nucleotide sequence) coding for G protein coupled receptor protein in a DNA library which comprises
xe2x80x83(i)
{circle around (1)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 1, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 3, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 5, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 6, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 7, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 10, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 14, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 16 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 18, and
{circle around (2)} at least-one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 2, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 4, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 8, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 9, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 11, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 15, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 17 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 19; or
xe2x80x83(ii)
{circle around (1)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 1 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 12, and
{circle around (2)} at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 13;
(107) a vector comprising the DNA according to the above (7);
(108) an expression system comprising an open reading frame (ORF) of DNA derived from a G protein coupled receptor protein DNA according to any of the above (7) and (42) to (57), wherein the ORF is operably linked to a control sequence compatible with a desired host cell;
(109) a transformant (including a transfectant) carrying a vector of the above (107) or an expression system of the above (108);
(110) a process for producing a G protein coupled receptor protein or a salt thereof, which comprises culturing the transformant of the above (109) to express said G protein coupled receptor protein on the membrane of the transformant;
(111) a method for expressing a polypeptide of G protein coupled receptor protein, comprising:
(a) providing a transformant of the above (59) or (109); and
(b) incubating the transformant under conditions which allow expression of the polypeptide of G protein coupled receptor protein;
(112) a method for preparing a transformant according to the above (59) or (109), comprising:
(a) providing a host cell capable of transformation;
(b) providing a vector according to the above (58) or (107) or an expression system according to the above (108); and
(c) incubating (a) with (b) under conditions which allow transformation of the host cell with the vector or the expression system;
(113) a pharmaceutical composition according to the above (82) or (88), comprising an effective amount of a compound according to the above (81) or (87) or a pharmaceutically acceptable salt thereof in admixture with a pharmaceutically acceptable carrier, excipient or diluent;
(114) the pharmaceutical composition according to the above (82) or (88), for inhibiting the binding of a G protein coupled receptor protein according to the present invention with a ligand;
(115) a method for inhibiting the binding of a G protein coupled receptor protein according to the present invention with a ligand in a medium which comprises contacting an effective amount of a compound according to the above (81) or (87) or a salt thereof with said medium;
(116) a method for modulating the activity of a G protein coupled receptor protein comprising contacting cells expressing the G protein coupled receptor protein with a an effective amount of a compound according to the above (81) or (87) or a salt thereof;
(117) the ligand according to the above (90) being labeled with a detectable reporter;
(118) the antibody according to the above (64) wherein the antibody is labeled with a detectable reporter;
(119) a pharmaceutical composition for controlling an expression of G protein coupled receptor protein, which comprises an effective amount of the antisense DNA according to the above (99), and
(120) a culture product produced by a transformant according to the above (59) or (109).
Yet another aspect of the present invention is
(121) a DNA according to the above (1) wherein the DNA is an oligonucleotide having from 8 to 60 base residues;
(122) a DNA according to the above (1) wherein the DNA is synthetic;
(123) a DNA (or nucleotide sequence) coding for a G protein coupled receptor protein or a fragment thereof, which is obtained through the method according to any of the above (5) to (32);
(124) a DNA (or nucleotide sequence) according to the above (123), wherein said G protein coupled receptor protein is selected from the group consisting of angiotensin receptor, bombesin receptor, canavinoid receptor, cholecystokinin receptor, glutamine receptor, serotonin receptor, melatonin receptor, neuropeptide Y receptor, opioid receptor, purine receptor, vasopressin receptor, oxytocin receptor, VIP receptor (vasoactive intestinal and related peptide receptor), somatostatin receptor, dopamine receptor, motilin receptor, amylin receptor, bradykinin receptor, CGRP receptor (calcitonin gene related peptide receptor), adrenomedullin receptor, leukotriene receptor, pancreastatin receptor, prostaglandin receptor, thromboxane receptor, adenosine receptor, adrenaline receptor, xcex1- and xcex2-chemokine receptor including IL-8, GROxcex1, GROxcex2, GROxcex3, NAP-2, ENA-78, PF4, IP10, GCP-2, MCP-1, HC14, MCP-3, I-309, MIP1xcex1, MIP-1xcex2, and RANTES receptors, endothelin receptor, enterogastrin receptor, histamine receptor, neurotensin receptor, TRH receptor, pancreatic polypeptide receptor, and galanin receptor; and
(125) a culture product produced by a transformant according to the above (59) or (109).
As used herein the term xe2x80x9csubstantial equivalent(s)xe2x80x9d means that the activity of the protein, e.g., nature of the ligand binding activity, and physical characteristics are substantially the same. Substitutions, deletions or insertions of amino acids often do not produce radical changes in the physical and chemical characteristics of a polypeptide, in which case polypeptides containing the substitution, deletion, or insertion would be considered to be substantially equivalent to polypeptides lacking the substitution, deletion, or insertion. Substantially equivalent substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. The non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine, The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartio acid and glutamic acid.